Erythropoietin (Epo) is a glycoprotein hormone of molecular weight 34 kilodaltons (kDa) that is produced in the mammalian kidney and liver. Epo is a key component in erythropoiesis, inducing the proliferation and differentiation of red cell progenitors. Epo activity also is associated with the activation of a number of erythroid-specific genes, including globin and carbonic anhydrase. Bondurant et al., Mol. Cell Biol. 5:675–683 (1985); Koury et al., J. Cell. Physiol. 126:259–265 (1986). The erythropoietin receptor (EpoR) is a member of the hematopoietic/cytokine/growth factor receptor family, which includes several other growth factor receptors, such as the interleukin (IL)-3,-4 and -6 receptors, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor as well as the prolactin and growth hormone receptors. Bazan, Proc. Natl. Acad. Sci USA 87:6934–6938 (1990). Members of the cytokine receptor family contain four conserved residues and a tryptophan-serine-X-tryptophan-serine motif positioned just outside the transmembrane region. The conserved sequences are thought to be involved in protein-protein interactions. Chiba et al., Biochim. Biophys. Res. Comm. 184:485–490 (1992).
EpoR cDNA has been isolated recently from mouse liver, Tojo et al., Biochem. Biophys. Res. Comm. 148: 443–48 (1987) and from human fetal liver. Jones et al., Blood 76:31–35 (1990); Winkelmann et al., Blood 76:24–30 (1990). The full length EpoR cDNA sequence is shown in the Sequence Listing as SEQ ID NO: 4. The human cDNA encodes a polypeptide chain of MW about 55 kDa and having about 508 amino acids. The polypeptide encoded by SEQ ID NO:4 is SEQ ID NO:5. Genomic clones of human EpoR have been isolated and sequenced. Penny and Forget, Genomics 11:974–80(1991); Noguchi et al., Blood 78:2548–2556 (1991). Analysis of the coding sequence predicts about 24 amino acid residues in a signal peptide, about 226 amino acids in an extracellular domain, about 23 amino acids in a membrane-spanning domain, and about 235 amino acids in a cytoplasmic domain. D'Andrea and Zon, J. Clin. Invest. 86:681–687 (1990); Jones et al., Blood 76:31–35, (1990); Penny and Forget, Genomics 11: 974–80 (1991). The mature human EpoR protein has about 484 amino acids. All human erythroid progenitor cells have been shown to contain Epo receptors. Binding of Epo appears to decline as erythroid progenitor cells mature, until Epo receptors are not detectable on reticulocytes. Sawada et al., J. Clin. Invest. 80:357–366 (1987). Sawada et al., J. Cell. Physiol. 137:337 (1988). Epo maintains the cellular viability of the erythroid progenitor cells and allows them to proceed with mitosis and differentiation. Two major erythroid progenitors responsive to Epo are the Burst-forming units-erythroid (BFU-E) and the Colony-forming units-erythroid (CFU-E). The Epo receptor number correlates very well with the response to Epo in normal BFU-E and CFU-E. Epo receptor numbers appear to decline after reaching the peak receptor number at the CFU-E stage in human and murine cells. Sawada et al., J. Clin. Invest. 80:357–366 (1987); Landschulz et al., Blood 73:1476–1486 (1989). The recovery of Epo receptors after removal of Epo appears to be dependent on protein synthesis, which suggests downregulation of Epo receptor by degradation, and the subsequent upregulation of receptors by the new synthesis of receptors when Epo is removed. Sawyer and Hankins, Blood 72:132 (1988). Studies of Epo receptors on megakaryocytes and erythroid progenitors suggest that there is a link between the regulation of erythropoiesis and thrombopoiesis, in that stimulation of cell division by both cell types is controlled by Epo receptor numbers. Berridge et al., Blood 72:970–977 (1988). Although the Epo receptor has been cloned, the precise mechanisms involved in binding of Epo to Epo receptors and the relationship to subsequent erythropoietic processes are not known.
Characterization of the Epo receptor (EpoR) has been difficult due to the extremely small quantities of EpoR that can be obtained from natural sources. Thus, the mechanism of Epo interaction with its receptor, which stimulates erythropoiesis, is still unknown. D'Andrea and Zon, J. Clin. Invest. 86:681–687 (1990). Recently this mechanism has been of great interest in understanding the role of growth factors and their receptors in leukemogenesis; altered hematopoietic growth factors and their receptors may contribute to tumorigenesis and leukemogenesis. Dunbar et al., Science 245:1493–1496 (1989); Li et al., J. Virol. 57:534–538 (1986).
Several studies of the correlation between the Epo responsiveness of a particular cell type and the affinity of the cell type for Epo have reported discordant results. These studies have used recombinant Epo or EpoR possessing some non-native amino acid sequence from the corresponding plasmid vectors. Berridge et al., Blood 72:970–977 (1988); Harris et al., J. Biol. Chem. 267: 15205–09 (1992). It is possible that tertiary structural changes and/or other features of these recombinant Epo or EpoR molecules have changed the characteristics of the native protein. Thus, it would be a significant advance to obtain substantially pure fragments of the Epo receptor, free of extraneous (e.g, vector) amino acid sequence. Although it could not be predicted whether or not such fragments would retain functional activity, nevertheless a purified extracellular domain fragment would be particularly useful since Epo binds to the extracellular domain of the Epo receptor.